Cell or drug encapsulation device having a wet seal

ABSTRACT

A biologically implantable containment device having a wet seal, the device being adaptable for drug formulations or cell suspensions. A porous membrane, in a tubular configuration, is formed and can be configured as part of a closed cell-tight system for loading. During loading, the containment device membrane is wet, while the loading system remains cell-tight. The containment device is wet-sealed through a combination of heat and pressure, while the system remains cell-tight. Sealing the containment device substantially or completely eliminates metabolic functioning of any organisms in the vicinity of the closure. The wet-seal is formed by melting a thermoplastic material that is in contact with the membrane. The containment device is separated from the cell-tight loading system, which remains closed after separation.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of co-pending application Ser. No.10/462,915, which is a division of application Ser. No. 09/515,264 filedFeb. 29, 2000, now U.S. Pat. No. 6,617,151.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to the field of implantable devices and moreparticularly to devices that are wet-sealed.

2. Background Information

Within the field of implantable devices, it is known to providepermeable membrane structures for implantation, the structuresconfigured to hold drug formulations or cellular suspensions. A numberof techniques have been proposed to form those structures and seal thestructures. In the majority of those known techniques, the device ismanufactured without the cellular suspension or drug formulation.Subsequent loading of the cellular suspension or drug formulation mayoccur outside a host or after the device is implanted into the host.When a suitable cell suspension or drug formulation is loaded into thedevice, it is typical and frequently desirable for the permeablemembrane to become wet with fluid. Given the nature of the membranes, itis known that sealing a wet membrane can be difficult or impossible.This is because known glues and solvents that are appropriate formembranes in a dry state are frequently not compatible with a wetmembrane, or are toxic to cell suspensions loaded into the membranestructure. To offset this difficulty, different dry and wet sealtechniques have been proposed.

In one technique, such as disclosed in U.S. Pat. No. 5,902,745 to Butleret al., the device includes a permeable tubular membrane, which issealed with a mechanical seal after loading the device with anappropriate cell suspension. In this technique, the membrane is wet whenthe seal is formed, but seal integrity relies on the quality of themechanical seal. With implantable devices, the mechanical sealdimensions are small and can be difficult to reliably manipulate. Inaddition, because the loading and sealing operations can be distinct,there is an opportunity for contamination of the device exterior withcells from the cell suspension after the loading operation.

In another technique, such as disclosed in U.S. Pat. Nos. 5,653,687;5,653,688; 5,713,887; 5,738,673 and 5,932,460 issued to Mills et al, adry seal is formed after the device is loaded. However, the loading andsealing steps are distinct and the device is open to the loadingenvironment after loading and before the device is sealed. For some ofthese seals, the seal depends on mechanical aspects of the seal. Some ofthe disclosed seal techniques require a solvent based seal. The solventsdescribed may be toxic to the cell suspension, however. In oneparticular embodiment of the seal, a portion of the device is broken offand removed after loading and prior to sealing. This action presents astrong possibility of contaminating the loading environment. Thiscontamination can be subsequently transferred to the exterior of thedevice, or to other devices or apparatus.

In another technique, such as disclosed in U.S. Pat. Nos. 5,545,223 and5,549,675 issued to Neuenfeldt et al., the apparatus or device is firstimplanted in a host and then loaded with a cellular suspension in thehost environment. In addition to problems that are described with wetsealing of the device, this technique is performed through an incisionor injection port following implantation in the host, thereby exposingthe device and the host to a risk of contamination. The technique ofNeuenfeldt et al. also requires a larger device to accommodate thedistance between the cell suspension and the seal. This larger devicealso produces greater host trauma during implantation. In some of theknown techniques, the device or apparatus is loaded in an area that isremote from the host. In these methods, the loading process or apparatusprovides opportunities for contamination from drug formulations or cellsuspensions between the loading and the sealing steps.

As described, the methods available do not provide a secure and reliableclosure system, that reduces the possibility of contamination duringloading. In addition, the methods available do not provide a method toreliably seal a device after the membrane is wet. Systems and methods toaddress these and other deficiencies are needed.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a method of closing acontainment device that comprises wetting at least a portion of apermeable polymeric membrane of the containment device with a liquid andapplying heat to at least a portion of a wetted thermoplastic polymer inassociation with the membrane to create a closure. Such a closure isreferred to herein as a “wet seal.” In this “wet sealing” process, thethermoplastic polymer melts at a lower temperature than the polymericmembrane. Once melted, the thermoplastic polymer integrates with thepolymeric membrane and flows along surfaces and into availableinterstices of the membrane. Through passageways become filled with themelted polymer, thereby blocking fluid communication in the polymericmembrane in the region of the closure. When the thermoplastic polymercools below its melt temperature, a closure is formed in the device. Theclosure is cell-tight and often liquid-tight. The portion of the devicehaving a closure formed with a wet seal delineates a cell-impermeableregion of the device.

The application of heat may be accompanied by slight pressure and a heatsink may be applied to limit heat transfer beyond the closure region tothe permeable membrane. After forming the closure, the method mayinclude pressure checking the closure integrity. The device may includeadditional closures that are formed by wet or dry sealing techniques.

In one aspect, the present invention provides a method of closing acontainment device that comprises wetting a porous expandedpolytetrafluoroethylene (ePTFE) membrane of the containment device witha liquid, and applying heat to a portion of the membrane incommunication with a thermoplastic polymer, such as fluorinated ethylenepropylene (FEP), to create a closure. The closure is formed by meltingand fusing of the polymer to itself and the membrane in the presence ofthe liquid.

In one aspect, the present invention provides a method of closing acontainment device comprising wetting a permeable membrane of thecontainment device with a liquid, wetting a thermoplastic polymer regionof the device with the liquid and applying heat directly to thethermoplastic polymer region to create a closure. In this aspect, thethermoplastic polymer region is joined to the permeable membrane beforewet sealing the containment device.

In one aspect, the present invention provides a method of closing acontainment device that comprises applying sufficient heat to a portionof a permeable membrane in association with a thermoplastic polymer tomelt and flow the thermoplastic polymer, followed by twisting themembrane/thermoplastic polymer combination in the region of the heatingto form a closure. The membrane/thermoplastic polymer combination isalso elongated while heating or twisting the materials. After heating,twisting, and elongation a separation region is formed and the membraneis cut in the separation region. In one aspect, the present inventionprovides a containment device comprising a membrane, a polymer incommunication with the membrane, and a closure. The closure is createdby applying heat to a portion of the membrane and a portion of thepolymer after wetting the membrane with a liquid.

In one aspect, the present invention provides a containment devicecomprising a membrane, a polymer region joined to the membrane and aclosure. The closure is created by applying heat directly to the polymerregion after wetting the membrane and the polymer region with a liquid.

In one aspect, the present invention provides a containment devicecomprising a membrane and a closure. The closure is created by applyingheat to a portion of the membrane and twisting the membrane in theregion of the heating. In one aspect, the present invention provides amethod of forming a containment device. The method comprises forming acontainment region that includes a membrane, forming a thermoplasticpolymer region joined to the membrane and forming a closure region. Theclosure region communicates with the containment region and applyingheat directly to the thermoplastic polymer region after wetting themembrane creates a closure in the closure region.

In one aspect, the present invention provides a method of forming acontainment device. The method comprises forming a containment regionthat includes a membrane, and forming a closure region. The closureregion communicates with the containment region and applying heat to aportion of the membrane and twisting the membrane in the region of theheating creates a closure in the closure region.

In one aspect, the present invention provides a method of loading acontainment device comprising placing a cell suspension or drugformulation in a containment region of the device through a closEableopening, the containment region including a membrane and the liquidwetting the membrane, and creating a closure in the closeable opening byapplying heat to a portion of the membrane in association with athermoplastic polymer.

In one aspect, the present invention provides a method of loading acontainment device comprising creating a closed cell-tight system, thesystem including the containment device and a source of metabolicallyactive cells. Loading a containment region of the device with themetabolically active cells via a closure region, the containment regionincluding a membrane. Creating a closure at the closure region, theclosure substantially or completely eliminating metabolically activecells in the vicinity of the closure, and subsequent separation of thesource of cells while maintaining a closed cell-tight system.

The foregoing specific aspects, objects and advantages of the inventionare illustrative of those which can be achieved by the present inventionand are not intended to be exhaustive or limiting of the possibleadvantages that can be realized. Thus, the aspects, objects andadvantages of this invention will be apparent from the descriptionherein or can be learned from practicing the invention, both as embodiedherein or as modified in view of any variations which may be apparent tothose skilled in the art. Accordingly the present invention resides inthe novel parts, constructions, arrangements, combinations andimprovements herein shown and described.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing features and other aspects of the invention are explainedin the following description taken in conjunction with the accompanyingfigures wherein:

FIG. 1 illustrates a containment device of the instant invention and animplantable containment apparatus suitable for holding the containmentdevice;

FIG. 2 illustrates a cross-section of an embodiment of the containmentdevice of the instant invention;

FIG. 3 illustrates an embodiment to form the tubular membrane of theinstant invention; FIG. 4 illustrates various cross-section embodimentsof the containment device of the instant invention;

FIG. 5 illustrates an embodiment of the containment device of theinstant invention;

FIG. 6 illustrates an embodiment of a dry seal on an end of thecontainment device of the instant invention;

FIG. 7 illustrates a cross-section of an embodiment of the containmentdevice of the instant invention;

FIGS. 7A-7C illustrate cross-sections of embodiments of the containmentdevice of the instant invention;

FIG. 8 illustrates an embodiment of a heat source used to form closureregions or seals in containment devices of the instant invention;

FIG. 9 illustrates an embodiment of steps to form a containment deviceof the instant invention;

FIG. 10 illustrates a loading hood used in one embodiment for loadingthe containment device of the instant invention;

FIG. 11 illustrates a loading jig in one embodiment for loading thecontainment device of the instant invention;

FIG. 12 illustrates an embodiment of a loading apparatus attached to acontainment device of the instant invention;

FIG. 13 illustrates an embodiment of loading cell suspension into aloading apparatus attached to a containment device of the instantinvention;

FIG. 14 illustrates an embodiment of loading cell suspension into acontainment device of the instant invention and clamping the containmentdevice after the cell suspension is loaded;

FIG. 15 illustrates an embodiment of heat sealing or closing acontainment device of the instant invention using an electrically heatedclamp;

FIG. 16 illustrates an embodiment of twisting and elongating the closureregion of the containment device of the instant invention;

FIG. 17 illustrates an embodiment of separating the sealed containmentdevice of the instant invention from the loading apparatus, and theresulting closure at the closure region;

FIG. 18 illustrates an embodiment of steps to close or seal acontainment device of the instant invention;

FIG. 19 illustrates alternative embodiments of the containment device ofthe instant invention;

FIG. 20 illustrates an alternative embodiment of the containment deviceof the instant invention; and

FIG. 21A illustrates an alternative embodiment of the containment deviceof the instant invention.

FIG. 22 is a graph showing the presence of chemical groups from residueassociated with wet seals of the instant invention.

It is understood that the drawings are for illustration only and are notlimiting. It is specifically understood that the scale of elements andrelative dimensions in the drawings may be exaggerated to provideclarity and illustrate individual aspects.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a containment device, methods of making thedevice and methods of loading and sealing the device. The containmentdevice is particularly suited for use as a medical device, such as acell encapsulation device, a drug delivery device, or a gene therapydevice. The containment device may be inserted into a previouslyimplanted containment apparatus residing within a recipient, such as ananimal or human, or it may be implanted directly in a recipient. Thedevice includes a permeable membrane, which partially defines anenclosed space of the device, and a closure region of the device.Materials (e.g., cells, or drugs) are loaded into the device from aloading device through the closure region into a containment region,after which the closure region is treated to form a closure. The closureis typically created by heating a thermoplastic polymer associated withthe permeable membrane in the presence of a liquid that wets at least aportion of the membrane in the closure region. The liquid may also wetat least a portion of the thermoplastic polymer. Wetting of the membraneis often a result of filling the device with a liquid, such as a cellsuspension or drug formulation, or from sterilization procedure prior tofilling. Forming the closure may include pressure or clamping as part ofthe sealing process. A closure formed in this manner is referred toherein as a “wet seal.” Different embodiments for creating the closureafter loading the containment device are disclosed. In one embodiment,the closure is created when heat is applied to a portion of a tubularporous expanded polytetrafluoroethylene (ePTFE) membrane, which includesa tube of fluorinated ethylene propylene (FEP) thermoplastic polymer inassociation with the ePTFE membrane. The applied heat is sufficient tomelt the FEP thermoplastic, but not of a magnitude to melt orsubstantially degrade the ePTFE membrane. While the FEP is in a meltedstate, the tubular membrane in the region of the melted FEP is pressedtogether, preferably by twisting the tubular device around itslongitudinal axis, and elongated, thereby creating the closure. Ineffect, the FEP and ePTFE form a fused or welded cell-free andcell-tight wet seal closure. After forming the wet seal closure, thecontainment device is separated from the cell loading device by a cutthrough the fused ePTFE/FEP material in the closure region.

After a wet seal is formed, thermally degraded residue from cells orcell culture media can usually be found on surfaces of material in theclosure region. Residue may also be embedded in material of the closureregion. The presence of such residue can be detected by sampling charredmaterial from the closure region and subjecting the sample and a controlto analysis. A preferred analytical method is Fourier Transform InfraredSpectroscopy (FTIR). In comparison with a control, chemical groupsindicating degradation of cells or cell culture media in the wet sealregion become evident as changes in peaks on a graph. An example of sucha graph is shown in FIG. 22. In the graph, the curve represented by adotted line is from a control sample. The curve represented by acontinuous line are peaks generated from a test sample. Differences inthe peaks on the graph are indicative of chemical changes in the cellculture media used to load cells into a device of the present inventionat the time of a thermally effective wet sealing process in the closureregion. Such residue is only present in the closure region if a wet sealhas been formed in the region.

Another important aspect of the invention is that the containment deviceand the loading device form a closed cell-tight system during loading,sealing and separation. This closed cell-tight aspect of the system ismaintained during the loading and closure or sealing and subsequentseparation of the containment device. In this manner, contamination ofthe device exterior by cells from either the loaded device or the celldelivery device is completely or substantially prevented.

During trials, containment devices were manufactured, loaded, sealed andlater implanted according to the instant invention. These testspositively demonstrate that the methods and apparatus disclosed in theinstant application accomplish the stated objectives of reducing oreliminating contamination of the device exterior and containing cellswithin the device. In vitro testing demonstrated that exteriorcontamination during device loading was reduced or eliminated by usingthe disclosed method and system. In vivo testing for up to 6 monthsshowed that cell containment within the containment device was possiblewithout device or seal failure. These test results provide strongevidence that containment devices, manufactured, loaded, sealed andimplanted according to the instant invention, will maintain the desiredisolation of cells within the device.

Another important aspect of the invention is that forming the closurethrough clamping or pressure does not compromise the mechanicalintegrity of the containment device.

Uses or Applications of the Device

The containment device according to the instant invention can bedirectly implanted in a recipient in need of treatments provided by thecontents of the device. When directly implanted in a recipient, knownsurgical techniques are used to prepare an implantation site andposition the containment device in the recipient. This includes anincision at the site and preparation of a tissue envelope to hold thecontainment device. For ease of insertion, incisions may be used toallow threading of the containment device between the incisions. Theimplantation site can be a subcutaneous location that is somewhatprotected from external forces and not subject to significant flexduring normal recipient activities. As examples, the inner forearm orthe inner upper thigh of a human may be appropriate sites.

The containment device can also be indirectly implanted in a recipientwith the used of a containment apparatus for the device. Such anapparatus is disclosed in U.S. Pat. No. 5,843,069, issued to Butler etal. (the '069 patent) and incorporated herein by reference. Referring toFIG. 1, the containment device 100 according to the instant inventioncan also be placed or replaced in an implantable containment apparatus120 of the '069 patent. The apparatus and methods disclosed in the '069patent overcome some of the above-described problems with directimplantation.

When the containment device 100 of the instant invention is used withthe implantable containment apparatus 120 of the '069 patent, thecontainment device is first prepared or filled with the appropriate cellsuspension or drug formulation. After prepared or filled, thecontainment device is wet sealed, as described in greater detail below.Once wet sealed, the containment device is placed or replaced in theimplantable containment device of the '069 patent.

In a preferred embodiment, the containment device provides a sealedenvironment for cellular suspensions or drug formulations. However, thedevice has other embodiments and applications where a permeable membraneencloses a space and the device is placed within a host or recipient.For example, as micro-machining techniques become more common, it willbe appropriate for the containment device 100 to contain amicro-miniature factory, the factory providing different forms ofprocessing. In this embodiment, the factory performs any number ofdifferent manufacturing functions. These include neutralizing orchanging the composition of molecules, substances or compounds in therecipient. The micro-miniature factory within the containment device ofthe instant invention can also produce drugs or compounds that areharvested from the host or recipient. The factory is passive, such as acatalyst, or it is active, with a power source such as internalmetabolism of proteins from the nutrient, or external power from outsidethe host or recipient. In short, the invention does not envisionlimiting the materials within the containment device to only cellsuspensions or drug formulations.

Materials for the Membrane of the Present Invention

The containment device of the instant invention includes a permeablemembrane. Preferred permeable membranes are much like the permeablemembrane of the '069 patent's implantable containment apparatus. Themembrane of the instant invention allows transport of nutrients,cellular wastes, and other materials through and across the membrane,but the membrane prevents cell movement or migration through and acrossthe membrane.

Referring to FIG. 2, the exterior tube 202 of containment device 100 ofthe present invention is made, primarily, of a permeable polymericmaterial having sieving properties. The sieving properties of suchpermeable polymeric materials can be adjusted to control passage ofsolutes, biochemical substances, viruses, or cells, for example, throughthe material, primarily on the basis of size. Preferred permeablepolymeric materials in the present invention are porous. In general, asthe average pore size of a porous polymeric material increases,increasingly larger biochemicals and biological entities are able topass through the material. In the present invention, porous polymericmaterials capable of preventing the passage of biological cells throughthe material, while permitting biological molecules to pass through thematerial, are most preferred.

Porous polymeric materials suitable for construction of the exteriortube 202 of a containment device of the present invention include, butare not limited to, porous expanded polytetrafluoroethylene (ePTFE);porous polypropylenes (PP), such as Celgard® (Celanese Separations,Inc., Charlotte, N.C.), Solvex® (Millipore Corporation, Bedford, Mass.),and Metricel® (Gelman Sciences, Ann Arbor, Mich.); porous polyethylene(PE), including stretched and sintered forms; porous polyvinylidenefluoride or PVDF (e.g., Durapore®, Millipore Corporation, Bedford,Mass., or FP VericelB®, Gelman Sciences, Ann Arbor, Mich.); track-etchedand other porous polycarbonates (e.g., Isopore®, Millipore Corporation,Bedford, Mass.); woven or non-woven collections of fibers or yarns, orfibrous matrices, such as those described by Fournier et al. in U.S.Pat. No. 5,387,237 (incorporated herein by reference); or foams ofpolyvinyl alcohol (PVA), polypropylene (PP), or polyethylene (PE),either alone or in combination.

Other materials suitable for construction of exterior tube 202 includepolymers such as biocompatible polyamides (FH-66®, Gambro AB, Lund,Sweden); cellulosics such as cellulose acetate, nitrocellulose, andmixtures thereof (e.g., NC®, Schleicher and Schuell, Inc., Keene, N.H.,and MF®, Millipore Corporation, Bedford, Mass., or Metricel®, GelmanSciences, Ann Arbor, Mich.); polyacrylamide and its copolymers withacrylic acid and acrylonitrile (e.g., Hypan®, Hymedix, Inc., Dayton,N.J.); polyacrylonitrile or PAN and its copolymers, including withsodium methallysulfonate (AN-69®, Hospal, Lyon, France) and poly(acrylonitrile-covinyl chloride) or (PAN-PVC); porous poly(ether etherketones) or PEEKs; porous polysulfones including poly (ether sulfones)or PESs (e.g., Tuffryn® or Supor®), Gelman Sciences, Ann Arbor, Mich.);or stable, strong, biocompatible hydrogels used in soft contact lenses,such as poly (2-hydroxyethyl methacrylate) or polyHEMA,poly(N-vinyl-2-pyrrolidone) or PVP, and mixtures and copolymers thereof.

Expanded, porous polytetrafluoroethylene is preferred for constructionof tube 202. Porous expanded polytetrafluoroethylene (ePTFE) ischaracterized as a material having void spaces defined by nodes andfibrils. Methods for making ePTFE are taught by Gore in U.S. Pat. Nos.3,953,566 and 4,187,390, each of which is incorporated herein byreference. However, the methods for making ePTFE are not subjects of thepresent invention.

For ePTFE, or similar fibrillated material, the pore size is related tothe fibril length and fibril density of the material and the thicknessof the material. In the present invention, appropriate ePTFE materialsare selected that resist cellular movement across the thickness of thematerial, while being selectively permeable to macromolecules. Thesematerials have microstructures (i.e., fibril length and fibril density)in combination with material thickness control in large part the sievingproperties, or permeability, of the membrane material. Another approachto characterize the sieving properties of a porous materials, such asePTFE, is to measure resistance to fluid flow across the materials. Oneappropriate measure is the pressure at which a gas can pass through theporous material and form bubbles when the material is immersed in anappropriate liquid. This measurement technique is referred to as a“bubble point” metric.

Method of Making the Device

Referring to FIG. 2, in a preferred embodiment, a containment device 100of the present invention is in the form of a tube. The tube walls createa central lumen 206, which can hold a drug formulation, cell suspension,or other agent. In a preferred embodiment with a cell suspension, lumen206 surrounds a central core 204, which can have a number ofcross-sectional shapes. Examples of the various cross-sectional shapesare illustrated in FIG. 4, with a star shape 401, a perforated cylinder403, a randomly porous matrix 405 and a bulb extrusion 407.

The tube walls 202 are made of a membrane, which comprises an ePTFEmaterial. The membrane is a layer of ePTFE material that is a very thin,very strong non-woven web composed substantially of tightly spacedfibrils in which there are essentially no nodes. The fibril lengths haveaverage dimensions ranging between about 0.2 and about 4.0 microns, asmeasured by photomicrography. The fibril density is observed to be veryhigh and tightly packed with multiple points of contact. The points ofcontact do not have sufficient polytetrafluoroethylene material to bereferred to as nodes. The thickness of the material in its finished formis between about 1 micron and about 25 microns. The preferred method ofmaking the membrane utilizes a portion of a method taught by Bacino inU.S. Pat. No. 5,476,589 entitled “Porous PTFE Film And A ManufacturingMethod Therefor,” which is incorporated herein by reference.

Referring to FIG. 3, a preferred method to form a containment.device ofthe present invention in a tubular shape is by wrapping the ePTFEmembrane 301, made in accordance with the teachings of Bacino, on amandrel 303. Longitudinal and helical orientations of the wrapped filmmay be used. A single layer or multiple layers of wrapped film may beemployed. The wraps may be set so as to overlap, or to gap, depending onthe setting of such variables as film width, wrap angle, and mandreldiameter. In many applications, the overlap is preferably about 50%.This construction is then heated from about 340° C. to about 400° C.,preferably 385° C., for about 5-10 minutes to bond the respective layersto each other.

Another way to form an ePTFE containment device of the present inventionis through direction extrusion and expansion of the membrane as a tube.However, the various methods to form a tubular membrane are not keyaspects of the invention. A tubular ePTFE membrane, formed as describedabove, is a hydrophobic membrane. Accordingly, the membrane does notreadily permit liquid water to enter and traverse the void or porousspaces and passages of the membrane. It is known to apply certainalcohols, low surface tension liquids, wetting agents or surfactants tothe ePTFE to render the membrane wettable with liquid water. Thesemethods are described in U.S. Pat. No. 5,902,745 to Butler et al., thedisclosure of which is incorporated herein by reference. However,methods to form a wettable membrane are not subjects of the presentinvention.

In a preferred embodiment, the membrane is treated to make it wettable.In this embodiment a very dilute aqueous solution of a wetting agent,such as polyvinyl alcohol (PVA) is used. For example, 0.001% polyvinylalcohol in saline (weight to volume or w/v) provides enough wettingagent to the ePTFE material to prevent, or limit, spontaneous dewettingof the material. This also prevents, or limits, evolution of air bubblesin a containment device loaded with cells. The PVA also renders thenormally opaque ePTFE material essentially translucent to transparentupon wetting. Suitable wetting agents and/or surfactants for use in thismethod include, but are not limited to, polyvinyl alcohol, polyethyleneglycol, sodium dodecyl sulfate, fluorosurfactants, pluronics, and bilesalts in percentages ranging from about 0.001-5.0%. Suitable solventsfor this method include, but are not limited to, saline, water, andaqueous buffers, for example.

In the preferred embodiment of the present invention, wetting agentsand/or surfactants are adsorbed onto the surfaces and into the voidspaces, pores, or passages of the ePTFE membrane and preferablyimmobilized in situ in order to make the ePTFE material wettable withliquid water. There are many ways to immobilize wetting agents orsurfactants, such as, cross-linking, substrate grafting, plasmaimmobilization, ionic complexation, and free radical grafting, etc. Inone example, cross-linking the adsorbed wetting agent or surfactant onthe ePTFE in situ immobilizes the wetting agent or surfactant on theePTFE material. Certain wetting agents or surfactants can be used thatrender ePTFE spontaneously and substantially completely liquid waterwettable. A spontaneously and substantially completely water wettableePTFE material permits liquid water to flow along the surface andthrough the passages of the material by merely contacting the materialwith liquid water. Suitable wetting agents or surfactants for use in thepresent invention include, but are not limited, to polyvinyl alcohol,poly(tetrafluoroethylene-co-vinyl alcohol), polyacrylic acid,polyethylenimine, and polyethylene glycol. Wetting agents and/orsurfactants are adsorbed in various ways, such as solution, or neat,adsorption, vapor deposition, plasma immobilization, and thin filmassembly, for example. Preferably, polyvinyl alcohol is adsorbed toePTFE by adsorbing the polyvinyl alcohol onto the surfaces and into theporous, or void, spaces of the material, followed by immobilization viacross-linking the polyvinyl alcohol to itself with a dialdehyde such asglutaraldehyde.

A membrane of the present invention made of water wettable ePTFE isstrong enough to withstand hydrostatic pressures sufficient to causewater to be forced through the pores of the material across thethickness of the membrane. When water is being forced across thethickness of the membrane, the water wettable ePTFE material functionsas a filter, or an ultrafilter, depending on the permeability of thematerial. As water moves, or seeps, across the thickness of themembrane, it tends to collect into droplets on the outer surface of themembrane. As adjacent droplets grow in size, they merge and run off ofthe cover. This process is referred to herein as “weeping.” Most waterwettable membranes of the present invention are sufficiently permeableto water for pressurized water to visibly weep from the membrane withoutgross channeling of water.

Ideally, the membrane of the present invention is sufficiently waterpermeable to allow the ready separation of aqueous fluid from cellsunder relatively low pressure. A ready weep flow of ranging from about0.01 ml/cm²/minute to about 100 ml/cm²/minute at a pressure ranging fromless than about 3.4*10⁴ Pa to about 6.9*10⁵ Pa should permit relativelyrapid cell concentration within the device.

This is an extremely beneficial attribute of the present invention.Unlike other cell containment devices that require cell concentrationbefore insertion of the cells in the cell device and then carefullycalculated and controlled transfer, the present invention allows cellsto be easily transferred to any desired concentration with minimalpre-concentration steps. Further, by flushing a cell-filled apparatusafter initial loading of the cells, a user can assure that all cells areflushed into the device and not left as a wasted residue on theapparatus.

Another benefit of the membrane becoming essentially translucent totransparent is that in a translucent or transparent condition, the cellsin the device can be observed through the cover both during and afterloading of cells. This not only assists in the loading of cells, butalso makes monitoring of the cells during use much easier. Additionally,position of various elements of the containment device are more visibleduring assembly when the membrane is translucent.

In addition to using cells suspended in aqueous fluids in the presentinvention, cells suspended in viscous fluids, such as alginate, can alsobe loaded into the invention. With these cell suspensions, much lessfluid weeps through the permeable membrane as the suspension is loadedin the device. This often requires the cell suspension to be moreprecisely characterized in terms of cells numbers than with the aqueousfluids described above. Islets of Langerhans are examples of cell typesthat often benefit from being suspended in a viscous fluid when used inthe present invention.

The permeable membrane of the present invention should prevent cellsfrom moving into or out or the device, but allow the passage ofnutrients, waste products, and bioactive substances secreted by cellscontained within the device. In one embodiment, the membrane excludesparticles on a molecular scale. Such molecular weight cut off (MWCO)properties may be useful for excluding proteins, etc., produced by theimmune system of a recipient from traversing the membrane that wouldadversely effect cells encapsulated in the device. The precise MWCOrange appropriate for a particular application will vary depending onthe membrane material, type of cells contained within the device, thesize of the therapeutic cell product to be released into the surroundingenvironment, and the host environment, etc. Accordingly, selectivelypermeable membranes having a MWCO of between about 10 kD to about 2000kD may be suitable for use in the present invention. A MWCO range ofbetween about 30 kD and 150 kD is particularly preferred in applicationswhere it is desired to isolate the contained cells from contact withmolecules of the immune system capable of recognizing or destroying thecontained cells.

Referring now to FIG. 5, a preferred method of making a containmentdevice is as follows. This description is particularly appropriate for adevice that will carry a cellular suspension. A central core 204 isplaced within a tubular membrane 202. The core is a biologically inertmaterial, such as silicone, and has a cross-sectional shape that isappropriate for the cell suspension. Examples of the cross-sectionalshapes that are appropriate for a cell suspension are provided in FIG.4. In a preferred embodiment, the core has a stellar cross-section 401.However, the cross-sectional shape of the core is not critical to theinstant invention and may be any of a number of different shapes, suchas those shown in FIG. 4. The core 204 length is only slightly shorterthat the length of a finished containment device. The tubular membrane202 is longer than the core 204 to allow closure of the membrane at oneend and attachment of a loading device at the opposite end of themembrane. In this embodiment, the core is not attached to the membranein the final device. In other embodiments, the central core is attachedto the membrane in the closure region.

Referring to FIG. 6, after core 204 is placed within the tubularmembrane 202, a polymer plug 506 is placed in one end of the tubularmembrane, abutting core 204. In a preferred embodiment, plug 506 isfluorinated ethylene propylene (FEP). The core, plug and membrane arearranged so that approximately 2 mm of membrane 202 extends beyond theFEP plug and the FEP plug abuts the silicone core. In this arrangement,a heat source or implement 602 is applied to the membrane in thevicinity of the FEP plug. The temperature of heat source 602 istypically between 350° C. and 450° C., preferably 390° C., which isabove the melt point of the FEP and causes the FEP to melt and flow. Thetemperature of the heat source is also slightly above the melt point ofthe ePTFE membrane. However, the heat exposure is not so great to causeany significant degradation or damage to the ePTFE membrane. As a resultthe membrane combines with the FEP plug to form a closure or seal 520 atthe distal end of the containment device. For purposes of distinguishingdifferent seals of the containment device, this seal will be termed adry seal. In one embodiment, the opening in heat source 602 is slightlysmaller than the diameter of FEP plug 506. In this embodiment, when heatsource 602 is applied, a slight clamping pressure helps to form dry seal520. After cooling, this closure or seal 520 is substantially orcompletely impermeable to cells that are eventually loaded in thedevice. The closure is also generally impermeable to liquids within thedevice. This closure is considered to be a dry seal, because it iscreated by a dry seal technique. In this context, the closure techniqueis dry because the ePTFE membrane, the silicone core in the vicinity ofthe closure region, and the FEP plug are dry when the closure is created(i.e., aqueous fluids are absent during the formation of the closure).

Referring to FIGS. 5 and 7, after forming the dry closure on one end ofthe containment device, a blunt tip, such as an 18 gage needle 508 about25 mm long is inserted into a tube 510 of a colored thermoplasticmaterial. In a preferred embodiment, the tube is a colored thermoplasticmaterial made of FEP about 25 mm long. Needle 508 and FEP tube 510assembly are inserted into the proximal end of the tubular ePTFEmembrane 202 that is opposite the dry seal 520.

The needle 508 and FEP 510 tube are advanced within the ePTFE membrane202 until there is a slight gap of about 2 mm between the end of the FEPtube 510 and the silicone core 204. This slight gap is important for thesubsequent wet loading and sealing of the containment device. At thesame time, the silicone core 204 abuts the closure 520 at the other end.In this arrangement, heat source 602 is applied to the end of themembrane 202 with a slight clamping pressure where it bonds or seals themembrane to the FEP tube 510 and also bonds or seals the FEP tube to theneedle 508. The colored FEP becomes visible through the membrane afterremoving the heat and allowing the seal to cool. This helps to visuallyconfirm a cell impermeable seal. Referring to FIG. 7, a seal 720 isformed when the ePTFE membrane, FEP tube, and needle are dry. Therefore,it is formed by a dry seal technique and is a dry seal.

FIGS. 7A-7C illustrate additional ways in which a thermoplastic polymermaterial 512 can be placed in closure region 514 for wet sealing. Celldelivery means 515 are also provided in the figures.

Referring to FIG. 8, the same heat device used to form closure 520 atthe distal end of the containment device can be used to create seal 720.The heat device 602 is an electrically heated clamp or forceps with acylindrical opening 802 between the clamping jaws to surround thecontainment device 100. The jaws are about 2-3 mm thick. This designallows application of heat around the circumference of the containmentdevice and formation of a cell impermeable seal or closure. The heatdevice need not be electrically heated. For example, an ultrasonicheating device or a radio frequency induction device can be used togenerate heat in the desired location and thereby melt or fuse the FEPthermoplastic.

When used in combination with a containment apparatus of the typedisclosed by Butler et al. in U.S. Pat. No. 5,843,069, it is oftendesirable to form the sealed ends of the device into a smooth regularshape, such as a hemisphere. Applying a heated mold having the desiredshape to the sealed end of the present invention is a preferred way ofreshaping the sealed end. Heat can be generated in the mold withinfrared energy, ultrasound, or radio frequencies.

The method of making the containment device of the instant invention hasbeen described with reference to the figures. Referring to FIG. 9, thatmethod is summarized as follows: At step 902, ePTFE membrane material,made in the manner described above, is applied to a silver-plated copper(SPC) mandrel that is approximately 1.5-2.5 mm in diameter, andapproximately 810 mm long. The first wrap is a longitudinal wrap of 0.5inch wide ePTFE, and provides longitudinal strength to the resultingtubular ePTFE membrane. The longitudinal wrap that is applied at step902 is overlapped at the tape edges to form the tubular shape.

At step 904, subsequent bias or helical wraps of ePTFE are applied overthe longitudinal ePTFE wrap. In a preferred embodiment, these bias wrapshave about 50% overlap of the ePTFE tape. Multiple bias wraps areapplied to the mandrel, with the wrap directions alternating for eachsuccessive layer. In a preferred embodiment, six (6) alternating layersof 0.5 inch wide ePTFE tape are applied to the mandrel. At step 906, theePTFE wrapped mandrel is placed into an oven at approximately 385° C.for approximately eight (8) minutes. This heat step allows the ePTFEwrap layers to bond, forming a tubular porous membrane. The temperatureand time are selected so that the resulting bond is of sufficientstrength, without loss of the desired porosity. At step 908, the tubularePTFE membrane is treated with a wetting agent, such as polyvinylalcohol. This treatment helps to ensure that the normally hydrophobicePTFE will easily wet and form a porous permeable membrane for thetransfer of soluble materials.

At step 910, the tubular ePTFE membrane and mandrel are removed from theoven and cooled. The ends of the SPC mandrel are clamped, and themandrel is stretched approximately 30%. The stretch of the mandrel necksdown and slightly reduces the mandrel diameter. This reduction indiameter of the mandrel allows easy removal of the ePTFE tubularmembrane from the mandrel.

At step 912, the ePTFE tubular membrane is cut or trimmed to the desiredlength. In one embodiment, the wrapped membrane is about 810 mm long,which is sufficient to produce four (4) individual containment devices,with an 180 mm long membrane.

At step 914, a silicone core is inserted into the trimmed ePTFE tubularmembrane. The core diameter is less than the diameter of the tubularmembrane, allowing the core to easily slide into the membrane: In apreferred embodiment, the core is approximately 160 mm long and theePTFE membrane is approximately 180 mm long.

At step 916, an FEP plug, about 2 mm long, and also smaller in diameterthan the ePTFE tubular membrane, is put into the distal end of the ePTFEtubular membrane. The FEP plug, silicone core and ePTFE membrane areadjusted so that only a slight length (1-2 mm) of ePTFE membrane extendbeyond the FEP plug and the silicone core is close to, or abutts the FEPplug.

At step 918, the properly adjusted, or configured plug, core andmembrane are heat sealed at the distal end. The heat seal isaccomplished with the previously described electrically heated forceps.After heat seal, the distal end has an appearance similar to closure520, illustrated in FIG. 7.

At step 920, an 18 gage blunt tip needle is inserted into a colored FEPtube. The inner diameter of the FEP tube is slightly less than the outerdiameter of the needle, to provide a snug sliding fit.

At step 922, the combined needle and FEP tube are inserted into theproximal end of the ePTFE tubular membrane. The FEP tube is advancedinto the membrane until it is about 2 mm from the end of the siliconecore. This slight gap is helpful during cell loading to allow the cellsuspension to flow around the silicone core.

At step 924, the properly oriented ePTFE membrane, FEP tube and needleare heat-sealed using the same electrically heated forceps to form seal720. This is a secondary seal. After step 924, the containment device iscomplete and ready for sterilization and subsequent loading.

This completes the method of making the containment device. Aftercompleted, the containment device can be checked for leaks and closureintegrity with any known type of leak detection, including a bubblepoint check. In a bubble point check, a containment device is lightlypressurized to determine if there is any leakage. An inadequate seal isrevealed by the evolution of bubbles from the sealed regions of thedevice. The completed containment device can also be sterilized by anumber of known techniques, including but not limited to chemicalsterilization and steam autoclave. Steam autoclave has an advantage ofwetting the membrane and displacing air within the containment devicewith sterile liquid.

Method of Loading the Device

A containment device 100, manufactured according to the above-describedmethod, is preferably loaded with cell suspensions or drug formulations.As described above, an important aspect of the instant invention is thecell-impermeable nature of the device. This aspect helps to ensure thatany implant cells within the containment device remain within thedevice. Assurance that there is no implant cell leakage from thecontainment device is important because the cells may be geneticallyengineered or not compatible with the host immune system. If the cellsin the suspension can reproduce, it may be desirable to limit thatreproduction to the interior of the containment device. For this reason,the cell-impermeable nature of the containment device, including themembrane and closures, is important.

The sealing mechanisms of the present invention help to ensure thatcontamination from implanted cells does not occur from a faulty seal ina cell containment device. In addition, the present invention also helpsto ensure that contaminating implant cells do not originate from theloading system or process. This is accomplished by eliminating any openpaths for the implant cells from a loading device to the exterior of thecontainment device during loading. This reduces or eliminates thepossibility that implant cells will become attached to the containmentdevice exterior. Elimination or reduction in overall possibility ofcontamination also prevents a possibility that the implant cells cancontaminate the loading area. This helps to ensure that implant cellsare not inadvertently transferred from the loading area to the exteriorof the containment device during routine handling.

In the present invention, one method to accomplish these objectives isto use a closed cell-tight loading system. If the implant cell loadingsystem remains closed, there is little or no possibility that implantcells can escape from the system and contaminate the loading area orexterior of the containment device.

FIG. 10 illustrates an embodiment for manually loading containmentdevice 100. It is understood that alternatively, the loading steps canbe performed in an automated facility. When the containment device willcontain a cell suspension, the empty sterilized containment device 100,immersed in sterile liquid 1002 is placed in a loading hood 1004. Theequipment and tools to load and seal the containment device are also inthe loading hood. Prior to placing the device in the loading hood, airis removed from the containment device and replaced by the sterileliquid. When the device is sterilized in liquid by steam sterilization,evacuation or removal of air in the device is a natural consequence ofthe sterilization. For other sterilization techniques that do notnecessarily remove air within the containment device, an air evacuationstep is generally desired.

As illustrated in FIG. 11, the containment device 100 is placed in acell-loading fixture 1102, which is part of a loading jig 1104. Thisprovides a stable platform for subsequent operations.

Referring to FIG. 12, within the loading hood, a loading device 1202 isconnected to the needle 508 of the containment device 100, such as by aluer lock. In the manual system, loading device 1202 is a 1 ml syringe.In an automated system, which is not illustrated, loading device 1202 ispart of an automated cell suspension handling and delivery system. Theconnection between loading device 1202 and needle 508 is cell-tight.Sterile water may be present in the device and the needle and may spillonto the device at the time the two components are connected. Generallyspeaking, it is important that cells are not introduced into theimmediate vicinity of the open needle hub 508 until the connectionbetween the two components is first made.

When connected, devices 100 and 1202 become a closed cell-tight system.In the manual system illustrated in FIG. 12, cells in suspension areextracted with a pipette 1204 from a cell transfer container 1206.

Referring to FIG. 13, the pipette 1206 with cell suspension is used toplace the cell suspension into the loading device 1202. It is importantthat cells are not allowed to leak or escape from pipette 1206 as thecell suspension is transferred. A plunger 1302 is placed into theloading device 1202, thereby closing the loading system and forming aclosed cell-tight system.

Alternatively, the loading device can be charged with cells at adifferent station and then transported to the device loading station.Nevertheless, the cell delivery portion of the loading station issterile at the point of contact with the containment device. Theconnection between the loading device and the containment device iscell-tight to ensure a closed system.

A manual loading system is illustrated in FIGS. 10-13, to clearly showthe various steps for attaching the containment device 100 to theloading device 1202 and then placing a cell suspension into the loadingdevice. However, using an open syringe, there is a remote possibility ofcontamination in the loading area if cells are inadvertently spilledduring the pipette transfer from the cell transfer device to the loadingdevice. Therefore, a preferred embodiment is a fully closed system,where the loading device includes suitable interlocks and valves toavoid even this remote possibility of contamination. Only after thecontainment device and loading device are connected, is there anyloading of cells into the containment device. There is no path for cellsto escape from the closed cell-tight system during loading. The onlypath for the cells is from the loading device to the interior of thecontainment device.

Alternatively, a process that permits cells to leak or escape during theloading process may be acceptable, provided the leakage is contained andisolated from the cell containment device.

Referring to FIG. 14, after the cell suspension is within the loadingdevice 1202, the cell suspension is loaded into containment device 100by a slight pressure. This pressure is supplied by manually depressingplunger 1302. As the cell suspension passes into the containment device,the suspension is concentrated. This concentration is a consequence ofthe porous nature of the treated ePTFE tubular membrane in containmentdevice 100. Cells are unable to pass through the membrane, but thesuspension fluid is able to pass through the membrane. This serves as asieving action by the ePTFE membrane and retains or filters the cellswithin the containment device, while allowing excess cell suspensionfluid 1402 to pass through or weep from the ePTFE membrane ofcontainment device 100.

Alternatively, as illustrated in FIG. 21, if membrane 2102 does notreadily permit liquid passage, end 2101 of the device is configured toact as a receptacle for receiving a fluid stream. Permeable membrane2106 permits passage of liquids therethrough, while preventing cellsfrom escaping the device. After the containment device is loaded, theend is then sealed 2108. This is termed a “primary seal” because theclosure is formed after loading the device, while liquids of the cellsuspension are contacting the membrane and sealing polymer.

Methods of Sealing the Device

Once the cell suspension is loaded into the containment device, a clamp1404 is applied to the containment device. Preferably, the clamp isapplied to a region of the tubular membrane containing the silicone coresuch that the very end of the core is captured by the clamping force.The clamp serves two purposes. One purpose is to serve as a heat sinkduring subsequent sealing operations. The other purpose is to provide amethod to hold the containment device during the sealing operations.

After the cells are loaded, the system remains a closed cell-tightsystem during closure or wet-sealing of the containment device.Referring to FIGS. 15 and 16, heat source 604 is applied with a slightpressure to a portion of the ePTFE membrane 202 in the containmentdevice 100 that is in communication with the FEP polymer tube 510.During heating, the device is simultaneously twisted and elongated toform a closure region 1602. Ideally, the twist is about 360 degrees, butany twist of greater than about 45 degrees helps to accomplish theobjective of constricting and compressing the closure.

The twisting and elongation serves to provide a visible separationregion. The elongation also ensures that when the containment device isseparated from the needle and loading device, a seal or closure remainson each side of the separation. This wet-seal in closure region 1602 istermed a primary seal.

The heating serves to cauterize the closure and kill any cells that maybe within closure region 1602. The elongation and twisting also providesa slight pressure in the area of the closure and serves to provide avisible separation region. The elongation also ensures that when thecontainment device is separated from the needle and loading device, aseal or closure remains on each side of the separation, thereby ensuringthat the system remains a closed cell-tight system; cell leakage isprevented from the closure. In a preferred embodiment, this elongationand closure is about 2 mm long, which can be visibly observed andreadily bisected.

After the closure is created by heating, elongation, and twisting, theclosure is allowed to cool. At this point, the containment device withthe loaded cells or drugs remains attached to the needle by the closure.However, the closure eliminates any fluid passage between the needle andthe containment device. This can be verified by slightly pressurizingthe needle side of the closure and ensuring that no additional weepemerges from the containment device. With an integral closure, thecontainment device can be separated from the needle, without any fear ofcell leakage from either the containment device, or the loading device.Any cells within the closure region were either killed outright by theheat closure, or rendered metabolically non-functional. The only stepremaining in the seal or closure step is to separate the containmentdevice from the needle.

Referring to FIG. 17, after the closure is formed, the containmentdevice 100 is separated from the needle elements 1702. Separation isperformed with scissors or a knife. Care is taken to make the separationin the middle of the closure region, thereby maintaining cell-tightseals after the separation. The resulting closure on containment device100 and needle elements 1702 include a fused or welded area with FEP 510and ePTFE 202.

The subsequent processing includes disposal of needle elements 1702 andother associated elements of the loading device.

If the closure of the containment device extends, it may be appropriateto trim excess material from the closure. This is important if theexcess material can contact the containment apparatus during placementor replacement of the containment device.

The primary seal just described is formed as a wet seal, because it isformed in a containment device that has been loaded with a cellsuspension, resulting in a wet membrane.

Even after the containment device is sealed or closed and separated orcut from the loading device, the system remains closed. The closuresubstantially or completely seals the closure region and the heat usedto create the closure helps to ensure that any cells within the closureregion are either killed, or have their metabolic capabilitiesdestroyed. This ensures that cells are not released into the loadingarea or sterile field, and also helps ensure that multiple cell loadscan be performed without risk of contamination from previous loadingoperations.

In the described embodiment, heat from an electrically heated clamp isused to form the closures. In other embodiments, the closure of thecontainment device is created by other forms of heat application, suchas ultrasonic welding or radio frequency inductive heating. The onlyrequirement for heat closure is that the heat be applied locally in theclosure region, and that it melt the thermoplastic polymer to allowformation of the closure, without compromising the integrity of theePTFE membrane.

In another embodiment, the closure of the containment device is createdby heating only the membrane material, without any thermoplastic. Theonly requirement is that the resulting closure provide the requireddegree of closure or cell-impermeability. In another embodiment, aclosure is formed by heating only a thermoplastic material previouslyattached to the permeable membrane material. As shown in FIG. 19C, theclosure is made with a thermoplastic material having a portion isattached to the permeable membrane and a portion that extends beyond thepermeable membrane to provide a port made solely of thermoplasticmaterial.

In another embodiment, the closure at the end of the containment devicethat is normally created by a dry seal (i.e., the distal end) before thedevice is loaded, is created by a wet seal technique. In thisembodiment, the membrane is wet when the closure at the distal end iscreated. The source of the wet membrane is not limiting and may be theresult of a cell suspension load, a drug load, a wetting agent or asterile solution.

The closure can also be created by non-heat methods, such as solvents orchemicals. In this embodiment, an important aspect of the closure isthat after the closure is formed, there is little or no possibility thatliving or viable cells remaining within the closure region. If thesolvent or chemical is toxic to the cells, then formation of the closureitself may be sufficient to ensure that the closure region is free ofviable cells. However, if the solvent or chemical is not toxic to thecells, then an additional step must be provided to ensure that any cellswithin the closure region are killed or rendered non-metabolicallyfunctioning. For example, it may be appropriate to use an ultravioletlight cured or activated compound or glue to create the closure. Thesecompounds or glue may not be toxic by themselves, but the UV cure may besufficient to render the closure region cell dead or metabolicallyfunctioning.

The method of filling and sealing the containment device of the instantinvention has been described above with reference to the figures.Referring to FIG. 18, the steps of filling and sealing are summarized.At step 1802, the ePTFE tubular membrane and needle containment deviceis sterilized. As indicated above, this is with any of a number ofdifferent techniques, though steam sterilization is preferred asinitially wetting the membrane of the device.

At step 1804, air is removed from the containment device and themembrane is wet, if not accomplished in the sterilization step.

At step 1806, the containment device is attached to the cell deliveryapparatus.

At step 1807, a cell suspension is transferred to the cell deliveryapparatus.

At step 1808, a closed cell-tight system is formed.

At step 1809, a cell suspension is infused into the containment devicefrom the cell delivery apparatus. During this step, the cell suspensioncan be concentrated, as the membrane serves as a sieve to allow excesssuspension fluid to weep from the containment device membrane.

At step 1810, after the cell suspension is infused, a heat sink isapplied to the containment device. The heat sink is positioned justdistal to the eventual closure region.

At step 1812, the FEP tube is moved within the membrane so as to abuttthe silicone core. As discussed above, there is a gap between thesilicone core and the FEP tube to assist with cell loading. Prior tosealing the containment device, this gap is preferably closed at step1812.

At step 1814, a heat source is applied to the ePTFE tubular membrane inthe vicinity of the closure region. The heat source is any of a numberof different types, with an electrically heated forceps a preferredembodiment.

At step 1816, while the heat source is maintained near the closureregion, the ePTFE membrane is both elongated and twisted to form theclosure. This combination of actions applies pressure in the vicinity ofthe closure and helps to ensure a good seal. The heat, elongation andtwisting provides a cell kill zone. It is possible that the ePTFEmembrane is only twisted, or only elongated to create the closure.However, a combination provides the best closure.

At step 1818, the membrane closure is allowed to cool after removing theheat source. As the FEP and ePTFE cools, it hardens to form the closure.

At step 1820, the containment device is separated from the cell deliveryapparatus at the closure region. This is accomplished by cutting at themid-point of the closure with a scissors or knife.

At step 1822, integrity of the seal at the cell delivery apparatus ischecked, such as by slightly pressurizing the apparatus and observingany leaks.

At step 1824, ends of the containment device are trimmed to remove anyirregularity or sharp features which might be problems during subsequentimplant.

At this point, the containment device has been loaded with a cellsuspension, and the device has been sealed to form a cell-impermeableregion at the closure regions. The only steps remaining are preparationfor implant and implantation of the containment device directly into arecipient or indirectly in an implantable containment apparatus.

In the previous description, one embodiment and configuration of thecontainment device has been used to illustrate the inventive aspects.FIG. 19 illustrates another embodiment of the instant invention. In FIG.19A, the tubular membrane 202, FEP tube 510 and needle 508 areconfigured as generally described above. When the containment device ismade, heat is applied to the ePTFE membrane, causing the FEP andmembrane to fuse or melt and thereby create a seal 1902 with needle 508.

In another embodiment, illustrated in FIGS. 19B and 19C, the membrane issealed to the FEP tube. However, the membrane does not extend into theclosure region and the closure is created with only FEP. The FEP may befurther connected in a cell-tight fashion, to other cell deliverycomponents (see FIG. 19C). Cell-tight methods of connection include heatwelds, luer locking, etc.

In an embodiment illustrated in FIG. 19C, the tubular membrane 202 isheat sealed to FEP tube 510, to form seal 1906. However, membrane 202 isnot directly sealed to needle 508. In the embodiment illustrated in FIG.19C, FEP tube 510 is heat sealed to needle 508 to form secondary seal1904. As illustrated in FIG. 19C, an additional seal is made duringcontainment device manufacture. In the subsequent loading of thecontainment device, it is clear that a closure in seal region 1906 willinclude both ePTFE and FEP. However, a closure in seal region 1908 willinclude only FEP. Depending on a number of factors, it may be desireableto use the embodiment illustrated in FIG. 19A for some applications, andthe embodiments illustrated in FIGS. 19B and 19C for other applications.

FIG. 20 illustrates an alternative embodiment where a dry seal 2004 isformed on the end of a string or “sausage-link” of multiple containmentdevices 2006. The devices are simultaneously loaded and individualclosures 2004 are formed between devices 2006. The individual devicesare then separated at the closures 2004 between the devices 2006. Thishas an advantage of volume processing.

FIG. 21 illustrates an alternative embodiment, which is particularlyadvantageous where membrane weeping is not desired or possible. In thisembodiment, membrane 2102 surrounds core 2100. An FEP plug 2103 isbetween core 2100 and terminal 2105. Terminal 2105 is connected tomembrane 2102 and is either a filter 2106, to allow concentration of acell suspension as described above, or a fluid receptacle (notillustrated) to capture cell or drug suspension after flow through thecontainment device. A wet seal in effected in closure region 2014.

After loading with the drug or cell suspension, a closure 2108 is formedin one of the manners described above for other embodiments. In thismanner, the containment device can be readily loaded even when membrane2102 does not or cannot weep.

It is also apparent, though not illustrated, that the tubular membraneneed not be outside the FEP tube. For example, the needle might beinserted into the tubular membrane and the FEP tube placed over both themembrane and needle.

Clinical success with any implantable device containing somatic cells,engineered somatic cells, or immortalized transformed cells must containthose transplanted cells for the life of the device. Many of theproposed populations of cells to be used for somatic cell therapy in theinstant invention are both motile and immortal. Keeping those migratorycell populations within the device is a critical design parameter.

In vitro load testing was conducted in order to rigorously measure theability to load containment devices of the present invention without anyexternal cell contamination. Possible sources of cell contamination onexternal surfaces of the devices include cells being ultra-filtratedthrough the ePTFE membranes, back washed out the proximal end of thedevice before the wet seal is done, pinholes, or simply erroneousexternal contamination of the devices during the load procedure.Prokaryotic cells were used in the tests rather than mammalian cellsbecause of their much smaller size and much faster growth rates. Use ofprokaryotic cells is believed to be a valid and extremely sensitivemeasure of external contamination from any source during loading of thedevice.

Cell containment devices having wet seals were made as described aboveand presented with a bacterial challenge by loading broth culturescontaining either of two types of bacteria, M. luteus or P. aeruginosa,into the devices. These organisms were chosen due to size, shape andmotility differences that might affect their ability to beultrafiltrated and/or migrate through the ePTFE membranes of the presentinvention. Also, the colony growth and broth culture characteristicsallow easy and quick identification of each type of organism.

During loading, the fluid that normally ultra-filters through thepermeable membrane of a containment device of the present invention wasaseptically collected drop by drop onto standard microbiologic cultureplates of tryptic soy agar (TSA). The bottoms of the plates were markedindicating the exact location of where each drop fell. Once the deviceswere filled with the particular bacteria and the ultrafiltrate samplescollected and marked, the devices were wet sealed and placed into brothculture at 37 degree's centigrade along with their respective cultureplates (ultrafiltrate samples). The ultrafiltrate collected from theloading of the test devices were negative for growth of both organismstested. No colonies were present on the agar plates in the regionsmarked to indicate where the ultrafiltrate drops landed on the agar.

When cell-permissive devices (i.e., intentionally made leaky) wereloaded with either M. luteus or P. aeruginosa and the ultrafiltratecollected onto sterile agar plates, there was rapid colony growth inexactly the spots where the drops of ultrafiltrate landed. The colonycolor (M. luteus is yellow) and morphology was consistent with theoriginal two organisms used and not a contaminant. In addition, thecolonies were sampled and sent out for identification. The externalresults confirmed the identifications as Pseudomonas spp. or Micrococcusspp.

Further studies were conducted by placing test devices and controldevices in an in vitro culture. Test devices for this study wereintentionally made to leak bacteria. Control devices were made accordingto the teachings of the present invention and were not made to leakbacteria. Following loading of the devices with one of the two strainsof bacteria, the test and control devices were placed into a tryptic soybroth (TSB) for culture. If a wet sealed containment device is notcell-tight, bacteria escape the device and bloom into a turbid culturewithin a matter of hours. The TSB media around the test devices becameturbid within a matter of hours for both species of bacteria tested. Incontrast, the culture media in which the control devices were culturedshowed no turbidity.

Although illustrative embodiments have been described herein in detail,it should be noted and will be appreciated by those skilled in the artthat numerous variations may be made within the scope of this inventionwithout departing from the principle of this invention and withoutsacrificing its chief advantages.

Unless otherwise specifically stated, the terms and expressions havebeen used herein as terms of description and not terms of limitation.There is no intention to use the terms or expressions to exclude anyequivalents of features shown and described or portions thereof and thisinvention should be defined in accordance with the claims that follow.

1. A cell containment device comprising: a permeable membrane delimitinga space for containing cells therein; a closure region comprising athermoplastic polymer in association with the permeable membrane andmeans for placing cells in said space through said closure region;wherein said closure region is closeable with a wet seal.
 2. Acontainment device comprising: a membrane; a polymer in communicationwith the membrane; and at least one closure, the closure created byapplying heat to a portion of the membrane and to a portion of thepolymer after wetting the membrane with a liquid.
 3. A device accordingto claim 2, further comprising a cell suspension within a space enclosedby the membrane.
 4. A device according to claim 2, further comprising adrug formulation within a space enclosed by the membrane.
 5. A deviceaccording to claim 2, wherein the membrane includes a polymer membrane.6. A device according to claim 2, wherein the membrane includes a porousPTFE membrane.
 7. A device according to claim 2, wherein the membranesubstantially surrounds the polymer.
 8. A device according to claim 2,wherein the membrane is substantially tubular.
 9. A device according toclaim 2, further comprising a closure on a first end of the tubularmembrane and the closure created by applying heat is on a second end ofthe tubular membrane.
 10. A device according to claim 2, wherein thepolymer includes FEP.
 11. A device according to claim 2, wherein thepolymer is distinct from the membrane.
 12. A device according to claim2, wherein the closure is substantially part of the polymer.
 13. Adevice according to claim 2, wherein the closure is substantiallyseparate from the membrane.